muscle
Huang, Y.
Citation
Huang, Y. (2006, September 26). Molecular dissection of the dysferlin protein complex in
skeletal muscle. Gildeprint Drukkerijen, Enschede. Retrieved from
https://hdl.handle.net/1887/4573
Version: Corrected Publisher’s Version
License: Licence agreement concerning inclusion of doctoral thesis in theInstitutional Repository of the University of Leiden
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Yanchao Huang1,Steven H.Laval2*,Alexandra van Remoortere3*,Jacques Baudier4,Chriselle Benaud4,Louise V.B.Anderson2,Volker Straub2,Andre Deelder3,Rune R.Frants1,Johan T.den Dunnen1,Kate Bushby2,Silvère M . van der M aarel1
1 Center for Human and ClinicalGenetics,Leiden University M edicalCenter,Leiden,The Netherlands
2 Institute of Human Genetics,InternationalCentre for Life, Newcastle-upon-Tyne,UK
3 Departmentof Parasitology,Leiden University M edicalCenter, Leiden,The Netherlands
4 INSERM EM I-104,DRDC CEA-Grenoble,France
Abstract
Mutations in dysferlin cause limb girdle muscular dystrophy 2B, Miyoshi myopathy and distal anterior compartment myopathy. Dysferlin is proposed to play a role in muscle membrane repair. In order to gain functional insight into the molecular mechanisms of dysferlin, we have searched for dysferlin-interacting proteins in skeletal muscle. By co-immunoprecipitation coupled with mass spectrometry, we demonstrate that AHNAK interacts with dysferlin. W e defined the binding sites in dysferlin and AHNAK as the C2A domain in dysferlin and the carboxyterminal domain of AHNAK by GST-pull down assays. As expected the N-terminal domain of myoferlin also interacts with the carboxyterminal domain of AHNAK. In normal skeletal muscle, dysferlin and AHNAK colocalize at the sarcolemmal membrane and costamere. In dysferlinopathies, reduction or absence of dysferlin correlates with a secondary muscle-specific loss of AHNAK. Moreover, in regenerating rat muscle, dysferlin and AHNAK showed a marked increase and cytoplasmic localisation, consistent with the direct interaction between them. Our data suggest that dysferlin participates in the recruitment and stabilization of AHNAK to the sarcolemma and that AHNAK plays a role in dysferlin membrane repair process. It may also have significant implications for understanding the biology of AHNAK-containing exocytotic vesicles, "enlargosomes", in plasma membrane remodelling and repair.
Key words: Dysferlin, AHNAK, protein interaction, dysferlinopathies, skeletal muscle regeneration
Introducti
on
Mutations in dysferlin are responsible for autosomal recessive limb girdle muscular dystrophy 2B (LGMD2B), Miyoshi myopathy (MM) and distal anterior compartment myopathy (DMAT), a group of myopathies with a wide range of phenotypic variability referred to as the dysferlinopathies [1-3]. Regardless of phenotype, dysferlinopathy patients typically have absent or severely reduced levels of dysferlin in skeletal muscle.
spermatogenesis factor fer-1 that mediates spermatid vesicles/plasma membrane fusion [4]. Dysferlin is widely expressed, and in skeletal muscle it is primarily found at the plasma membrane [5] and in cytoplasmic vesicles [6]. Myoferlin is also expressed at the plasma membrane, and in addition found at the nuclear envelope. Dysferlin and myoferlin contain six C2 domains which are typically involved in the calcium-dependent binding to phospholipids and the most amino-terminal C2 domain, C2A, shows calcium-dependent phospholipid binding in vitro [7]. Various proteins have been suggested to interact with dysferlin including annexins A1 and A2, caveolin 3, calpain 3 (CAPN3), affixin (ȕ-parvin) and the dihydropyridine receptor (DHPR) [8-13] .
Although dysferlin is not considered an integral component of the dystrophin glycoprotein complex (DGC), patients with mutations in the DGC often show some reduced or altered dysferlin expression [6]. Dysferlin-null mice develop a slowly progressive muscular dystrophy with vesicular accumulations and sarcolemmal disruptions similar to those observed in human dysferlinopathy, and the vesicular accumulations are particularly prominent during skeletal muscle regeneration. Isolated myofibers from dysferlin null mice show impaired resealing following high-intensity laser irradiation, implicating dysferlin in some aspect of calcium dependent membrane repair [14].
By means of phage-display, we recently reported the successful selection of Llama-derived heavy chain antibody (HCAb) fragments specific for dysferlin [11]. We demonstrated these HCAb fragments to be functional in several immunological techniques including immunoprecipitation. Using immunoprecipitation followed by mass spectrometry we demonstrate here for the first time that AHNAK, a protein implied in cell membrane differentiation, repair and signal transduction [15-17], is in complex with dysferlin in skeletal muscle. GST-pull down assays and co-immunofluorescence microscopy provide further support for a direct interaction between dysferlin and AHNAK, as does the secondary reduction in skeletal muscle labelling for AHNAK in muscle from dysferlinopathy patients. We also found that dysferlin and AHNAK relocalize in a rat model of muscle regeneration, providing further evidence for a functional relationship between both proteins.
Materials and methods
C2C12 mouse cell lines were maintained in Dulbecco's modified Eagles medium with phenol red (Gibco BRL) supplemented with 10% fetal calf serum (FCS) (Gibco BRL), sodium pyruvate (final concentration 1 mM, Gibco BRL) and penicillin-streptomycin (100 IU/100 UG/ML, Gibco BRL). The cells were cultured in an incubator with 10% CO2 at 37qC.
Cells were induced to differentiate and fuse at 30-50% confluency by switching to serum-deprived medium (4% horse serum in Dulbecco's modified Eagles medium). After differentiation for 7 days, cells were harvested, resuspended in lysis buffer 1 (50 mM Tris, pH 7.4, 150 mM NaCl, 0.15% CHAPS (3-[3-cholamidopropyl]dimethylammonio)-1-propanesulfonic acid)) 1× protease inhibitor cocktail (Roche Molecular Biochemicals, Basel, Switzerland), and incubated on ice for 10 min. Cellular debris was removed by brief centrifugation at 8,000 g for 5 min at 4qC, and the protein concentration of the supernatant was assayed with by SDS-PAGE.
Recombinant protein production
pcDNAI/Amp eukaryotic expression vectors containing N-AHNAK1 (N-DY1) (residues 2-252), M-DY1 (residues 821-1330), C-DY1 (residues 4646-5643) were a kind gift from Dr. Takashi Hashimoto (Keio University School of Medicine, Tokyo, Japan). DNA fragments encoding the protein fragments N-DY1, M-DY1, and C-DY1, respectively, were obtained by BamHI/XhoI restriction from pcDNAI/Amp and ligated in the prokaryotic expression vector pET28a-GST (modified from pET28a (Novagen, Madison, WI) with an additional GST tag). Subsequently, pET28a-GST-C-DY1 was split in two parts by BamHI/SacI digestion (designated C1-DY1,
from residue 4646-5145) and by SacI/XhoI digestion (designated C2-DY1,
from residue 5146-5643) and ligated in BamHI/SacI-digested, or SacI/XhoI-digested pET28c-GST, respectively.
To generate the carboxyterminus of AHNAK2, C2-DY2 (aa 5146-5637), a
To generate DYSF C2A (aa 1-130), a 392 bp fragment was PCR amplified from a plasmid that contains entire human Dysferlin coding sequence with forward primer (5'- ATC GGG ATC CAT GCT GAG GGT CTT CAT C-3') and reverse primer (5'- ATC GCT CGA GCA CAG CTC CAG GCA GCG G-3') and cloned into the BamHI/XhoI-digested pET28a. DYSF2 (aa 2-1080) was generated by the plasmid that contains entire human dysferlin coding sequence with EcoRI/XhoI and ligated in the EcoRI/XhoI-digested prokaryotic expression vector pET28a. DYSF1 (aa 2-245) and DYSF3 (aa 1666-1788) were generated as previously described [11]. C2D (aa 1152-1285) and C2Q (aa 1314-1476) were generated in vitro using a TNT Coupled Rabbit Reticulocyte Lysate System as directed by the manufacturer (Promega).
Recombinant myoferlin proteins MYOF1 (aa 2-245), MYOF2 (aa 1-130) and MYOF C2A (aa 1-85) were produced by PCR amplification of 740 bp, 407 bp, 272 bp fragments from cDNA clone (clone ID: DKFZp686C16167Q2, RZPD), respectively. MYOF1 was generated with forward primer (5'-GGG AAT TCC TGC GAG TGA TTG T-3') and reverse primer (5'-GGG AAG CTT AAA CAA CTC ATC-3'); MYOF2 with forward primer (5'-GGGAATTCATGCTGCGAGTGA-3') and reverse primer (5'-GGGAAGCTTTGGAGCAGAAG-3'); and MYOFC2A with forward primer (5'-GGGAATTCATGCTGCGAGTGA-3') and reverse primer (5'-GGGAAGCTTAGTCGCCGTG-3'), respectively, and cloned into the TOPO 2.1 vector (Invitrogen). Subsequently, the fragments were digested with EcoRI/SalI, EcoRI/HindIII, and EcoRI/HindIII, respectively, and ligated in the prokaryotic expression vector pET28a (Novagen, Madison, WI). All constructs were sequence-verified (LGTC, Leiden, The Netherlands).
minutes, and then centrifuged at 500×g for 5 minutes. The supernatant was removed and the Glutathione-Sepharose was washed three times with binding buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 0.2% Triton X-100). The GST/GST-fusion protein bound beads were ready for pull-down assay. Antibodies
The antibodies used in this study were as follows. The monoclonal anti-dysferlin antibody NCL-Hamlet (Novocastra, Newcastle, UK) was used in a dilution of 1:300 for Western blot analysis and at a dilution of 1:50 or 1:100 for immunofluorescent microscopy. Monoclonal antibodies against ȕ-dystroglycan, dystrophin, ȕ- and Ȗ-sarcoglycan, and spectrin were as previously described [18]. Monoclonal antibody against caveolin-3 (Becton Dickinson) was used at a dilution of 1:1,000. Mouse monoclonal CD31 antibody (Abcam, Cambridge, UK) was diluted 1:500 for immunostaining. Mouse monoclonal antibody against DHPR (Abcam, Cambridge, UK) was diluted 1:200 for immunostaining. Secondary antibodies goat anti-mousealexa488 (Molecular Probes, Eugene, OR), goat anti-rabbitalexa594 (Molecular Probes), sheep anti-mouse (Amersham), swine anti-rabbit (DAKO) and rabbit anti-mouseHRP (DakoCytomation, Glostrup, Denmark) were diluted 1:250, 1:1,000, 1:500, 1:200 and 1:1,000, respectively. Affinity purified KIS/AHNAK polyclonal antibodies were used in appropriate dilutions as previously [19]. Mouse anti-T7 HRP (Novagen) was diluted 1:15,000 for Western blot analysis.
Co-immunoprecipitation and Western blotting
Mass spectrometric analysis and protein identification
Immunoprecipitated proteins were separated on SDS-PAGE. The protein bands were excised from sypro-stained gel and trypsin digested as described [20]. Briefly, minced gel pieces were first washed with H2O and acetonitril,
reduced with dithiothreitol at 56qC for 45 min, and then alkylated by iodoacetamide in the dark for 30 min. The gel was incubated in 30 Pl of a 5 ng/ul modified trypsin solution in 50 mM ammoniumbicarbonate, pH 8.6 and incubated at 37 qC o/n. The digests were acidified with aqueous TFA to a final concentration of 0.1% and the peptides were extracted with one change of 50 mM ammoniumbicarbonate. The sample was desalted and concentrated with a 10 Pl ZipTip C18 (Millipore, Bedford, MA), following the instructions provided by the manufacturer. Peptides were eluted with 1.5 Pl D-cyano 4-hydroxycinnamic acid matrix (0.33 mg/ml in Aceton/Ethanol (1:3) onto the MALDI target plate. Mass spectrometry (MS) and tandem mass spectromtery (MS/MS) data were aquired with the MALDI-TOF-TOF mass spectrometer (Ultraflex TOF/TOF mass spectrometer, Bruker Daltonics, Bremen, Germany). Proteins were identified by peptide mass fingerprint (PMF) and MS/MS peptide sequencing with the searching program MASCOT (http:\\www.matrixscience.com). NCBInr was the database used with the following search parameters: a mass tolerance of 0.1 Dalton and one misscleavage were allowed and peptide mass changes due to carbamidomethylation of cysteine and oxidation of methionine were taken into account.
Pull down assays
dyferlin protein fragments. After separation, proteins were transferred to PVDF membranes and dried o. n. Then, blots were incubated with NCL-hamlet (1: 300) for 2 h at RT and rabbit anti mouse HRP (1:2,000) for 1 h at RT for detection of endogenous dysferlin; anti-T7HRP antibody for the detection of recombinant dyferlin protein fragments. ECL plus was used for visualization.
Immunohistochemistry
For immunohistochemical examinations, transverse or longitudinal muscle cryosections of 5 µm thickness were fixed in 3.7% formaldehyde containing 0.1% triton X for 30 min, following by preincubation with phosphate-buffer saline containing 4% skimmed milk at room temperature for 2 h. The sections were next incubated with primary antibody fragments overnight at 4˚C, and subsequently with affinity-purified secondary antibodies at room temperature for 40 min, following by incubation of fluorescein-labeled tertiary antibody for 40 min at RT. Background staining was performed by omitting the primary antibody from the first step. The sections were washed with PBS, dehydrated with 70, 90, 100% ethanol and mounted in a DAPI (50 ng/µl)/ VectashieldTM mounting medium (Burlingame, CA). Final preparations were analysed with a Leica DMRA2 fluorescence microscope and images were obtained using Leica CW4000 digital system.
Regenerating rat muscle
(Perkin-Elmer). Images were collected and analysed on a Zeiss LSM-510 confocal microscope.
Results
Identification of AHNAK as a novel binding partner of dysferlin by co-immunoprecipitation and mass spectrometry
In order to identify novel protein partners for dysferlin, we performed immunoprecipitation followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Normal human muscle homogenates were immunoprecipitated with anti dysferlin HCAb fragment F4 and restained proteins were resolved on SDS-PAGE. The sypro-stained protein bands were excised and digested with trypsin and subjected to MS. A number of proteins co-precipitated with dysferlin by both peptide mass fingerprinting and MS/MS peptide sequencing (data not shown). The searching program MASCOT was used to identify the protein species. A most interesting novel component of the IP-complex was AHNAK, which was chosen for further characterization.
To confirm that AHNAK was co-immunoprecipitated with dysferlin, independent IPs were analyzed by Western blotting with affinity purified polyclonal antibodies raised against AHNAK [19]. As shown in Fig. 1B, multiple protein bands corresponding to different AHNAK protein isoforms were detected in the IP fraction with HCAb fragment F4, but not in the IP fraction of unrelated HCAb fragments or in the IP fraction without primary HCAb fragments. This specific immunoreactivity indicates an interaction between dysferlin and AHNAK.
Interaction of GST AHNAK fusion proteins with endogenous dysferlin
Two homologues of AHNAK exist in the human and mouse genome: AHNAK1 localized at human chromosome 11 and AHNAK2 at human chromosome 14 [21]. Both proteins are composed of a large number of highly conserved repeat segments but differ in their C-terminal domain. The polyclonal AHNAK antisera react to both proteins and cannot discriminate them.
-DY1: aa 5146-5643) were applied in a GST-pull down assay with human muscle (Fig. 2B) or mouse C2C12 protein extracts (Fig. 2C). Western blot analysis of the pull down fractions using the dysferlin-specific monoclonal antibody NCL-Hamlet showed that only the most carboxyterminal end of AHNAK1, C2-DY1, was able to bind to dysferlin. No binding was observed
for equivalent amounts of the GST fusion proteins N-DY1, M-DY1, C1-DY1 or for the control unfused GST protein.
In C2C12 cells, we identified two protein bands reactive to NCL-Hamlet, a prominent high molecular weight isoform and a less prominent low molecular weight isoform of dysferlin. While the low molecular weight isoform is most abundant in the precleared protein fraction, in our pull-down assay from C2C12 cells, only the high molecular weight isoform of dysferlin was pulled down by GST-C2-DY1, but not the low molecular
weight isoform (Fig. 2C).
Figure 1: Western blot analysis of co-immonoprecipitation by the selected HCAb fragments F4 from human muscle homogenates.
Panel A: Coimmunoprecipitated proteins were detected with NCL-hamlet anti dysferlin antibody. Dysferlin was immunoprecipitated by HCAb fragments F4 (lane 1) with Protein A Sepharose and detected with NCL-Hamlet. As controls, an unrelated HCAb fragment (lane 2) and Protein A Sepharose without HCAb fragment (lane 3) were included. Lanes 4 and 5 represent immunodetection of dysferlin in total muscle homogenates of human (lane 4) with NCL-Hamlet and without primary antibody (lane 5) as negative control.
Figure 2: Identification of the interaction site of AHNAK1 with endogenous dysferlin. (A) Schematic representation of GST-AHNAK1 fusion proteins used in GST-pull down assay. The interaction ability of the fusion proteins with endogenous dysferlin is indicated in the left. Western blot analyses of GST pull-down fractions using NCL-Hamlet to detect dysferlin. Human muscle (B) or C2C12 (C) cell lysates were incubated with GST (lane 1), GST fusion N-DY1(lane 2), GST fusion M-DY1 (lane 3), GST fusion C1-DY1 (lane 4), and GST fusion C2-DY1 (lane 5). Bound proteins were resolved on
SDS-PAGE, blotted on PVDF membrane and were probed by NCL-Hamlet. Lanes 6, 7 and 8 represent immunodetection of dysferlin in precleared human or C2C12 lysates (lane 6), total muscle homogenates of human or mouse (lane 7) and without primary antibody as negative control (lane 8). Only GST fusion C2-DY1 is able to pull down endogenous dysferlin while other fusion proteins were
negative. A molecular weight marker is indicated on the left.
Interaction of GST AHNAK-C2 with T7-tagged dysferlin fusion proteins
In order to determine whether both AHNAK proteins can bind dysferlin, whether this interaction is direct or indirect, and to further define the interaction domain of dysferlin, we produced HIS- and T7-tagged fusion proteins representing different domains of dysferlin including C2A (aa 2-130), C2D (aa 1152-1285) and C2Q (aa 1314-1476), as well as the dysferlin protein fragments DYSF1 (aa 2-245), DYSF2 (aa 1666-1788), and DYSF3 (aa 2-1080). After incubation with the C-terminal GST-AHNAK1 fusion construct GST-C2-DY1 or control GST protein pre-immobilized to
dysferlin (C2A, DYSF1 and DYSF3) were specifically pulled down by GST-C2-DY1 (Fig. 3 A-C). No binding was observed for equivalent
amounts of the fusion proteins lacking the C2A domain of dysferlin (DYSF2, C2D, C2Q; data not shown) and the control GST protein. Furthermore, a C-terminal GST-fusion construct of AHNAK2 (GST-C2
-DY2: aa 5146-5637), was also able to pull down dysferlin fragments incorporating the C2A domain of dysferlin. This interaction of dysferlin with AHNAK2 seemed even stronger than with AHNAK1. Therefore, this observation indicates that the carboxyterminal domains of both AHNAK proteins specifically and directly associate with C2A-dysferlin. A scheme in Fig. 3G represents the direct interaction of AHNAK proteins with dysferlin. Interaction of GST AHNAK-C2 with T7-tagged Myoferlin fusion proteins
To elucidate whether AHNAK can also interact with myoferlin, a similar pull-down strategy was performed by replacing the T7-tagged aminoterminal constructs of dysferlin with those of myoferlin. By analogy to dysferlin, there is a direct interaction of the N-terminal myoferlin with the C–terminal domains of both AHNAK proteins (Fig. 3 D-F). In general, the interaction between myoferlin and AHNAK2 was weaker than for AHNAK1 and as shown in Fig. 3F, only GST AHNAK1-C2 effectively
pulled down T7-tagged C2A-Myoferlin. A scheme in Fig. 3H represents the direct interaction of AHNAK proteins with myoferlin.
sections and both AHNAK and dysferlin colocalizes with DHPR in longitudinal sections of skeletal muscle.
Figure 3: Identification of the interaction site of dysferlin (A, B, C) and myoferlin (D, E, F) with C2
-DYs. GST fusion C2-DYs or unfused GST lysates were used in pull-down assays with lysates of
T7-tagged fusion proteins representing different domains of dysferlin including C2A (aa 1-130) (C), and the dysferlin protein fragments DYSF1 (aa 2-245) (A), DYSF2 (aa 2-1080) (B); MYOF C2A (aa 1-85) (F), and the myoferlin protein fragments MYOF1 (aa 2-245) (D), MYOF2 (aa 1-130) (E) as described in Materials and Methods. Bound proteins were separated by SDS-PAGE and anaylzed by immunoblotting with anti-T7HRP. In A-F, lanes 1-5 represent uninduced fusion proteins, induced fusion proteins, soluble fusion proteins, precleared fusion proteins and GST C2-DY1 pull-down
fractions, respectively. Lane 6 in A, B, F and lane 7 in C, D, E represents GST alone pull-down fractions. Lane 6 in C, D, and E represent GST C2-DY2 pull-down fractions. As shown, GST-C2-DY1
pulled down T7-tagged dysferlin fusion proteins in which the C2A domain was present in the fusion protein. Similarly, GST C2-DY2 precipitated tagged DYSF C2A, tagged MYOF1 and
T7-tagged MYOF2. These results demonstrated a specific and direct interaction between C2-DYs and
Figure 4: Immunofluorescent analysis of AHNAK and dysferlin. Double immunofluorescent analyses of AHNAK and dysferlin in normal human skeletal muscle cross sections showed that AHNAK is preferentially localized in the sarcolemma (4A). Double immunostaining for AHNAK and CD31 revealed a colocalization of AHNAK and CD31 in the blood vessel endothelia (4B). Double immunofluorescent analyses of AHNAK and dysferlin in longitudinal normal human skeletal muscle sections showed the colocalization of AHNAK and dysferlin at the sarcomere (4C) while double immunostaining for AHNAK and DHPR also revealed colocalization (4D).
Immunostaining of dysferlin and AHNAK on patient muscle sections
the sarcolemma in the absence of dysferlin, its presence in the blood vessels persists (Fig. 5B) suggesting a muscle-specific reduction of AHNAK in these patients.
Along with the analysis of dysferlinopathy patients, we also performed a random labelling of seven additional non-dysferlin muscular dystrophies with dysferlin and AHNAK antibodies. These patients had genetically confirmed diagnoses of BMD, FSHD, DMD, Į-sarcoglycanopathy, ȕ-sarcoglycanopathy, Ȗ-ȕ-sarcoglycanopathy, and LGMD1C (Fig. 5C). Both dysferlin and AHNAK showed normal expression at the sarcolemma in all cases except LGMD1C. In this case, dysferlin and AHNAK showed a secondary overall reduction along with the primary loss of caveolin-3. In this case, the residual AHNAK staining was also restricted to the blood vessels (Fig. 5B).
Immunostaining of dysferlin and AHNAK on regenerating mouse skeletal muscle sections
Figure 5: Immunofluorescent analysis of frozen muscle sections of three unrelated LGMD2B patients showed a comparable and similar reduction of AHNAK and dysferlin, with some patchy trace staining, at the sarcolemma of these three dysferlinpathy patients (Fig. 5A). The presence in the blood vessels persists (Fig. 5B) suggesting a muscle-specific reduction of AHNAK in these patients. Double staining was performed on serial sections. The right panels show the integrity of the membrane in the patients using anti-dystrophin and sarcoglycan antibodies.
Discussion
Dysferlin is a mammalian homologue of a Caenorhabditis elegans protein that mediates spermatid vesicle/plasma membrane fusion [4]. Dysferlin is ubiquitously expressed, with highest expression in skeletal muscle and heart [5]. Dysferlin-null mice progressively develop pathological characteristics of muscular dystrophy. In dysferlin-null mice there are sub-sarcolemmal accumulations of yet uncharacterised vesicles and dysferlin-deficient muscle fibers are defective in Ca2+-dependent sarcolemma resealing [14]. In order to gain further insights into the molecular mechanisms of dysferlin function, we have searched for proteins that interact with dysferlin in skeletal muscle. In this paper we describe a novel interaction of dysferlin with AHNAK based on IP coupled to MALDI-MS analysis, further confirmed by co-immunoprecipitation and GST pull-down assays, and by immunofluorescence microscopy analysis. CAPN3, whose mutations cause LGMD2A, has been confirmed to interact with dysferlin in a similar way [11]. Caveolin-3, affixin and annexins, another three known dysferlin interacting proteins, were undetectable due to their locations in the smears caused by the cross-reactivity of the secondary antibody against co-immunoprecipitated IgG molecules from the muscle homogenate.
AHNAK or has undergone a conformational change that impairs the binding between both proteins.
AHNAK is a family of two proteins (AHNAK1 and AHNAK2) of exceptionally large size (approximately 600-700 kDa) and characterized by common amino-acid sequences and structural features [21]. The AHNAK proteins have a tripartite structuring including the amino-terminal 251 amino acid large head, a large central region of 4390 amino acids composed of twenty-six repeated elements, and the carboxyl-terminal tail of 1002 amino acids. Like dysferlin, high expression levels of AHNAK are observed in all muscular cells including cardiomyocytes and skeletal muscle cells [19]. The uniform distribution of AHNAK at the plasma membrane of skeletal muscle is also observed in normal human skin [23] and mouse keratinocytes [24]. However, structure prediction algorithms do not provide evidence for the presence of transmembrane domains in AHNAK to explain the plasma membrane localization. It was suggested that its membrane association is probably dependent on specific interactions with the annexin A2/S100A10 complex in human epithelial MCF-7 cells through the AHNAK C-terminus [25]. Nevertheless, annexin A2/S100A10 complexes do not contain transmembrane domains either. Dysferlin has also been shown to associate with the annexin 2/S100A10 complex in a Ca2+- and membrane injury-dependent manner [12]. We thus propose a model in which the sarcolemmal localization and stabilization of AHNAK can be controlled by its direct interaction with dysferlin and myoferlin, which are anchored to the sarcolemma through their single transmembrane domain (Fig. 7).
Figure 7: A scheme representing the proteins involved in the dysferlin complex at sarcolemma in skeletal muscle. In the multimeric complex, known direct interactions include those between the beta2 subunit of the cardiac L-type Ca2+ channel and AHNAK, and between AHNAK and
annexin/S100A10. The interactions of dysferlin with CAPN3, caveolin-3 and annexin A1/A2, respectively, are either direct or indirect. In this study, we demonstrated the direct interaction between dysferlin and AHNAK, and between myoferlin and AHNAK, respectively (indicated with red lines). In the dysferlin complex, dysferlin, caveolin-3 and CAPN3 have been linked to LGMD 2B, LGMD 2A and LGMD 1C, respectively.
In addition, we have provided further evidence for a functional cooperation between dysferlin and AHNAK during muscle regeneration. Dysferlin and AHNAK show a marked increase and cytoplasmic localisation during regeneration, consistent with the direct interaction between them and mobilization of the AHNAK-dysferlin complex during repair and regeneration. Regulation of cellular AHNAK content in relationship with cell membrane remodelling and specialization has already been observed in epithelial and endothelial cells [25]. In these cells AHNAK stabilization is dependent on its recruitment to the plasma membrane through interaction with partner proteins, including the annexin2/S100A10 complex [25]. Dysferlin has also been shown to associate with the annexin 2/S100A10 complex in a Ca2+- and membrane injury-dependent manner [12]. Thus, we suggest that at the sarcolemmal membrane dysferlin, AHNAK and the annexin2/S100A10 may act in a single functional complex, including caveolin-3, affixin and CAPN3, with a role in renewal of the complex structures of the sarcolemma.
disruption and is thought to be involved in both plasma membrane differentiation and repair [16]. In this line, an interaction of dysferlin and AHNAK in striated muscle is particularly intriguing as dysferlin was already implied in patch fusion repair. Like the uncharacterised vesicular accumulation in SJL-Dysf mice, AHNAK-positive enlargosomes are concentrated in the cytoplasmic rim below the plasmalemma [16]. It is therefore imperative to further investigate the role of enlargeosomes in muscle membrane maintenance and repair.
Acknowledgments
We are grateful to Dr. Takashi Hashimoto, Keio University School of Medicine, for providing us with cDNA clones of human AHNAK. This work was supported by grants from SenterNovem (IOP-Genomics IGE01019) and the National Institutes of Health (NIH-NIAMS R21-AR48327-01).
Conflict of Interest statement: No conflicts of interest.
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